The long range goal of the proposed research is to understand basic mechanisms involved in the regulation of nuclear gene transcription. The yeast gal system is being studied. Three galactose system specific proteins, Gal3p, Gal80p, and Gal4p work to regulate the activation and repression of gal gene transcription. Gal4p is a DNA binding transcriptional activator whose activity is blocked by a particular association with Gal80p. In response to galactose, a Gal3p inducing signal alters the Gal80p-Gal4p association, resulting in Gal4p's ability to activate transcription through engagement with the general transcriptional macro-assembly (GTM). The overall goal is to determine the nature of Gal4p's engagement with the GTM, and how this is controlled by Gal3p's inducing signal acting to alter the Gal80p-Gal4p interplay. One line of proposed research aims to determine the significance of phosphorylation(s) and dephosphorylation(s) of the Gal4p protein that appear tightly correlated with Gal4p's engagement and disengagement with the GTM. The relevant phosphorylation sites in Gal4p will be determined by in vivo (32)P-labeling, phosphopeptide mapping, and site-directed mutagenesis. Once relevant sites are identified, and mutated, the altered Gal4p will be tested in vivo and in vitro for altered activities. The kinase(s) responsible will be identified by either 1) co- immunoprecipitation with Gal4p, 2) analysis of the activities of purified known kinases on Gal4p in vitro, 3) analysis of the effects of cloned kinase genes on Gal4p phosphorylations in vivo, or 4) genetic selections and screens to identify mutants defective in the Gal4p kinase(s). In other expts, ts mutants in PolII and TBP and the polymerase inhibitor thiolutin will be used to map the execution point of Gal4p protein kinase relative to TBP and PolII execution points in the transcription activation pathway. Gal3p will be tested for association with Gal80p and/or Gal4p using both in vitro and in vivo physical approaches. Mutagenesis and genetic selections will be carried out aimed at identify regions of Gal3p required for inducing activity, identifying novel Gal3/c, activated alleles for use as new probes of the Gal80p-Gal4p interplay, and identifying possibly new Gal3p-dependent inducing components. The proposed lines of research should generate new and useful information regarding the Gal3p-Gal80p-Gal4p induction mechanisms and provide tools for further work. Transcriptional regulatory mechanisms are prominent determinants of development, differentiation and adaptation in all organisms. Transcriptional regulation gone awry is a determinant of many human diseases, including some cancers. Our studies here with the typical eucaryote, yeast, should contribute to an understanding of transcriptional regulatory mechanisms in general, since the overall process and many, if not most, of the essential components have been conserved from yeast to man.